Inactivation of aldophosphamide by human aldehyde dehydrogenase isozyme 3

Tumors resistant to chemotherapeutic oxazaphosphorines such as cyclophospha mide often overexpress aldehyde dehydrogenase (ALDH), some isozymes of whic h catalyze the oxidization of aldophosphamide, an intermediate of cyclophos phamide activation with formation of inert carboxyphosphamide. Since resist ance to oxazaphosphorines can be produced in mammalian cells by transfectin g them with the gene for human ALDH isozyme 3 (hALDH3), it seems possible t hat patients receiving therapy for solid tumors with cyclophosphamide might be protected from myelosuppression by their prior transplantation with aut ologous bone marrow chat has been transduced with a retroviral vector causi ng overexpression of hALDH3. We investigated whether retroviral introductio n of hALDH3 into a human leukemia cell Line confers resistance to oxazaphos phorines. This was examined in the polyclonal transduced population, that i s, without selecting our high expression clones. hALDH3 activity was 0.016 IU/mg protein in the transduced cells (compared with 2 x 10(-5) IU/mg in un transduced cells), but there was no detectable resistance to aldophosphamid e-generating compounds (mafosfamide or 4-hydroperoxycyclophosphamide). The lack of protection was due, in part, to low catalytic activity of hALDH3 to wards aldophosphamide, since, with NAD as cofactor, the catalytic efficienc y of homogeneous, recombinant hALDH3 for aldophosphamide oxidation was show n to be about seven times lower than that of recombinant hALDH1. The two po lymorphic forms of hALDH3 had identical kinetics with either benzaldehyde o r aldophosphamide as substrate. Results of initial velocity measurements we re consistent with an ordered sequential mechanism for ALDH1 but not for hA LDH3; a kinetic mechanism for the latter is proposed, and the corresponding rate equation is presented. BIOCHEM PHARMACOL 60;3:325-338, 2000. (C) 2000 Elsevier Science Inc.